Cardiac Relaxation Research Articles

A reverse stroke characterizes the force generation of cardiac myofilaments, leading to an understanding of heart function

Changes in the molecular properties of cardiac myosin strongly affect the interactions of myosin with actin that result in cardiac contraction and relaxation. However, it remains unclear how myosin molecules work together in cardiac myofilaments and which properties of the individual myosin molecules impact force production to drive cardiac contractility. Here, we measured the force production of cardiac myofilaments using optical tweezers. The measurements revealed that stepwise force generation was associated with a higher frequency of backward steps at lower loads and higher stall forces than those of fast skeletal myofilaments. To understand these unique collective behaviors of cardiac myosin, the dynamic responses of single cardiac and fast skeletal myosin molecules, interacting with actin filaments, were evaluated under load. The cardiac myosin molecules switched among three distinct conformational positions, ranging from pre- to post-power stroke positions, in 1 mM ADP and 0 to 10 mM phosphate solution. In contrast to cardiac myosin, fast skeletal myosin stayed primarily in the post-power stroke position, suggesting that cardiac myosin executes the reverse stroke more frequently than fast skeletal myosin. To elucidate how the reverse stroke affects the force production of myofilaments and possibly heart function, a simulation model was developed that combines the results from the single-molecule and myofilament experiments. The results of this model suggest that the reversal of the cardiac myosin power stroke may be key to characterizing the force output of cardiac myosin ensembles and possibly to facilitating heart contractions.

SERCA2a stimulation by istaroxime improves intracellular Ca2+ handling and diastolic dysfunction in a model of diabetic cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): This work was supported by CVie Therapeutics Limited (Taipei, Taiwan) and Windtree Therapeutics (Warrington, USA) Diabetic cardiomyopathy is a multifactorial disease characterized by an early onset of diastolic dysfunction (DD) that precedes the development of systolic impairment. Mechanisms that can restore cardiac relaxation improving intracellular Ca2+ dynamics represent a promising therapeutic approach for cardiovascular diseases associated to DD. Istaroxime has the double property to accelerate Ca2+ uptake into sarcoplasmic reticulum (SR) through the SR Ca2+ pump (SERCA2a) stimulation and to inhibit Na+/K+ ATPase (NKA). The project aims to characterize istaroxime effects at a concentration (100 nM) marginally affecting NKA, in order to highlight its effects dependent on the stimulation of SERCA2a in a model of mild diabetes. Streptozotocin (STZ) treated diabetic rats were studied at 9 weeks after STZ injection in comparison to controls (CTR). Istaroxime effects were evaluated in vivo and in left ventricular (LV) preparations. STZ animals showed 1) marked DD not associated to cardiac fibrosis, 2) LV mass reduction associated to reduced LV cell dimension and T-tubules loss, 3) reduced LV SERCA2 protein level and activity and 4) slower SR Ca2+ uptake rate, 5) LV action potential (AP) prolongation and increased short-term variability (STV) of AP duration, 6) increased diastolic Ca2+, 7) unaltered SR Ca2+ content and stability in intact cells. Acute istaroxime infusion (0.11 mg/kg/min for 15 min) reduced DD in STZ rats. Accordingly, in STZ myocytes istaroxime (100 nM) stimulated SERCA2a activity and blunted STZ-induced abnormalities in LV Ca2+ dynamics. In CTR myocytes, istaroxime increased diastolic Ca2+ level due to NKA blockade albeit minimal, while its effects on SERCA2a were almost absent. SERCA2a stimulation by istaroxime improved STZ-induced DD and intracellular Ca2+ handling anomalies. Thus, SERCA2a stimulation can be considered a promising therapeutic approach for DD treatment. Abstract Figure.

Mathematical Modeling of Delayed Relaxation in Heart Failure with Preserved Ejection Fraction (HF‐pEF)‐ an Effect or a Cause?

The pathophysiological mechanisms underlying heart failure with preserved ejection fraction (HF-pEF) remain incompletely understood. One proposed hypothesis is that prolonged pressure decay of left ventricle during isovolumic relaxation, due to loss of enhancement of the ventricle, may lead to elevation of left ventricular end-diastolic pressure (LVEDP) and a reduction in stroke volume in HF-pEF subjects. However, while impaired relaxation is commonly observed in HF-pEF, it is unclear whether it is a contributing cause of HF-pEF or perhaps a consequence of other impairments. We sought to evaluate whether direct impairment in cardiac relaxation can contribute to a state of HF-pEF using a mathematical modeling approach. We utilized an existing mathematical model of cardio-renal hemodynamics in which a rectified sinusoidal twitch shape function drives left ventricular contraction and relaxation. To evaluate the effect of impaired relaxation, we altered the falling side of the twitch shape function so that isovolumetric relaxation time (IVRT) increased from 100 ms to 150 ms. In addition, we also evaluated the effect of delayed relaxation coupled with other ventricular changes associated with HF-pEF, including increased ventricular stiffness. Our simulations showed that when IVRT was increased, left ventricular pressure (LV pressure) fell more slowly during diastole. However, there was minimal effect on LVEDP. Pulmonary venous pressure also fell more slowly during diastole, and the peak pulmonary venous pressure during systole was slightly increased. Ejection fraction (EF) was minimally changed. The simulated presence of hypertension, hypertrophy, and/or increased cardiac stiffness did not impact the effect of slowed relaxation. While experimental validation is needed, our simulations suggest that impaired relaxation alone may not be a primary cause for increased preload experienced by many HF-pEF patients. It is possible that delayed relaxation is a consequence of other contributing mechanisms (e.g. impaired bioenergetics, calcium handling, or fibrosis which may impact ventricle stiffness and contractility) rather than a direct cause for elevated LV preload. While impaired relaxation had minimal effect on LVEDP, the small increases in peak pulmonary venous pressure and slower fall during diastole could impact pulmonary and atrial function in HF-pEF.

Combined Risk Factors Induce T Cell‐Mediated Diastolic Dysfunction in a Novel Mouse Model of Heart Failure with Preserved Ejection Fraction (HFpEF)

Heart Failure with Preserved Ejection Fraction (HFpEF) is a prevalent and heterogenous cardiovascular syndrome characterized by impaired cardiac relaxation (diastolic function) with preserved contractility (systolic function), generally as a result of the presentation of several comorbidities. Despite its high and increasing prevalence, there are no therapies available to directly treat or cure HFpEF. Obesity and hypertension, the two dominant risk factors of HFpEF, have been independently associated with T cell inflammation, however, the inflammatory mechanisms driving HFpEF by these two comorbidities together, and T cell contributions, remain elusive. We hypothesized that T cell immune responses and cardiotropism promote diastolic dysfunction and HFpEF. C57/BL6 (wild-type, WT) and T cell receptor alpha-deficient (Tcra-/-) male mice were fed a high fat diet (HFD) with L-NAME-treated water (0.5 g/L) for 5 weeks to mimic obesity and hypertension, respectively, a recently established model of HFpEF. Age- and sex-matched mice were fed standard chow (STD) as controls. Cardiac function was assessed using hemodynamic analyses, and plasma was collected to evaluate systemic levels of pro- and anti-inflammatory cytokines. The heart, mediastinal lymph nodes, inguinal lymph nodes, and spleen were harvested to characterize cardiac and systemic immune cell populations by flow cytometry. Cardiac tissue was also collected for gene expression analysis by qPCR of markers of adverse cardiac remodeling and HFpEF. WT mice fed HFD/L-NAME had increased cardiac CD11b+ cell abundance and increased CD4+ T cell infiltration compared to controls. Furthermore, in contrast to control mice, HFD/L-NAME-treated mice had an increased abundance of CD44hi/CD62Llo effector T cells in the mediastinal lymph nodes and spleen. This was associated with increased passive chamber stiffness (end-diastolic pressure volume relationship, EDPVR) and decreased expression of the unfolded protein response (UPR) proteins spliced form of X-box binding protein 1 and binding immunoglobulin protein (XBP1s and BIP), hallmarks of HFpEF. Additionally, HFD/L-NAME induced a 2.5-fold increase in plasma levels of IL-17, without altering the levels of interferon-gamma or IL-2. Strikingly, Tcra-/- mice did not develop diastolic dysfunction when fed HFD/L-NAME, supporting a causal role of T cells in HFpEF pathology. We conclude that T cell activation and recruitment to the heart contribute to diastolic dysfunction in HFpEF. Further studies will interrogate the mechanisms by which combined risk factors prime T cells for cardiac infiltration and effector function in the heart to induce and drive HFpEF.

SERCA2a stimulation by istaroxime improves intracellular Ca2+ handling and diastolic dysfunction in a model of diabetic cardiomyopathy.

Aims Diabetic cardiomyopathy is a multifactorial disease characterized by an early onset of diastolic dysfunction (DD) that precedes the development of systolic impairment. Mechanisms that can restore cardiac relaxation improving intracellular Ca2+ dynamics represent a promising therapeutic approach for cardiovascular diseases associated to DD. Istaroxime has the dual properties to accelerate Ca2+ uptake into sarcoplasmic reticulum (SR) through the SR Ca2+ pump (SERCA2a) stimulation and to inhibit Na+/K+ ATPase (NKA). This project aims to characterize istaroxime effects at a concentration (100 nmol/L) marginally affecting NKA, in order to highlight its effects dependent on the stimulation of SERCA2a in an animal model of mild diabetes.Methods and results Streptozotocin (STZ) treated diabetic rats were studied at 9 weeks after STZ injection in comparison to controls (CTR). Istaroxime effects were evaluated in vivo and in left ventricular (LV) preparations. STZ animals showed (i) marked DD not associated to cardiac fibrosis, (ii) LV mass reduction associated to reduced LV cell dimension and T-tubules loss, (iii) reduced LV SERCA2 protein level and activity and (iv) slower SR Ca2+ uptake rate, (v) LV action potential (AP) prolongation and increased short-term variability (STV) of AP duration, (vi) increased diastolic Ca2+, and (vii) unaltered SR Ca2+ content and stability in intact cells. Acute istaroxime infusion (0.11 mg/kg/min for 15 min) reduced DD in STZ rats. Accordingly, in STZ myocytes istaroxime (100 nmol/L) stimulated SERCA2a activity and blunted STZ-induced abnormalities in LV Ca2+ dynamics. In CTR myocytes, istaroxime increased diastolic Ca2+ level due to NKA blockade albeit minimal, while its effects on SERCA2a were almost absent.Conclusions SERCA2a stimulation by istaroxime improved STZ-induced DD and intracellular Ca2+ handling anomalies. Thus, SERCA2a stimulation can be considered a promising therapeutic approach for DD treatment.

Cardiac T-Tubule cBIN1-Microdomain, a Diagnostic Marker and Therapeutic Target of Heart Failure.

Since its first identification as a cardiac transverse tubule (t-tubule) protein, followed by the cloning of the cardiac isoform responsible for t-tubule membrane microdomain formation, cardiac bridging integrator 1 (cBIN1) and its organized microdomains have emerged as a key mechanism in maintaining normal beat-to-beat heart contraction and relaxation. The abnormal remodeling of cBIN1-microdomains occurs in stressed and diseased cardiomyocytes, contributing to the pathophysiology of heart failure. Due to the homeostatic turnover of t-tubule cBIN1-microdomains via microvesicle release into the peripheral circulation, plasma cBIN1 can be assayed as a liquid biopsy of cardiomyocyte health. A new blood test cBIN1 score (CS) has been developed as a dimensionless inverse index derived from plasma cBIN1 concentration with a diagnostic and prognostic power for clinical outcomes in stable ambulatory patients with heart failure with reduced or preserved ejection fraction (HFrEF or HFpEF). Recent evidence further indicates that exogenous cBIN1 introduced by adeno-associated virus 9-based gene therapy can rescue cardiac contraction and relaxation in failing hearts. The therapeutic potential of cBIN1 gene therapy is enormous given its ability to rescue cardiac inotropy and provide lusitropic protection in the meantime. These unprecedented capabilities of cBIN1 gene therapy are shifting the current paradigm of therapy development for heart failure, particularly HFpEF.

Compartmentation of β2 -adrenoceptor stimulated cAMP responses by phosphodiesterase types 2 and 3 in cardiac ventricular myocytes.

In cardiac myocytes, cyclic AMP (cAMP) produced by both β1 - and β2 -adrenoceptors increases L-type Ca2+ channel activity and myocyte contraction. However, only cAMP produced by β1 -adrenoceptors enhances myocyte relaxation through phospholamban-dependent regulation of the sarco/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2). Here we have tested the hypothesis that stimulation of β2 -adrenoceptors produces a cAMP signal that is unable to reach SERCA2 and determine what role, if any, phosphodiesterase (PDE) activity plays in this compartmentation. The cAMP responses produced by β1 -and β2 -adrenoceptor stimulation were studied in adult rat ventricular myocytes using two different fluorescence resonance energy transfer (FRET)-based biosensors, the Epac2-camps, which is expressed uniformly throughout the cytoplasm of the entire cell and the Epac2-αKAP, which is targeted to the SERCA2 signalling complex. Selective activation of β1 - or β2 -adrenoceptors produced cAMP responses detected by Epac2-camps. However, only stimulation of β1 -adrenoceptors produced a cAMP response detected by Epac2-αKAP. Yet, stimulation of β2 -adrenoceptors was able to produce a cAMP signal detected by Epac2-αKAP in the presence of selective inhibitors of PDE2 or PDE3, but not PDE4. These results support the conclusion that cAMP produced by β2 -adrenoceptor stimulation was not able to reach subcellular locations where the SERCA2 pump is located. Furthermore, this compartmentalized response is due at least in part to PDE2 and PDE3 activity. This discovery could lead to novel PDE-based therapeutic treatments aimed at correcting cardiac relaxation defects associated with certain forms of heart failure.

Smoking flies: testing the effect of tobacco cigarettes on heart function of Drosophila melanogaster.

ABSTRACTStudies about the relationship between substances consumed by humans and their impact on health, in animal models, have been a challenge due to differences between species in the animal kingdom. However, the homology of certain genes has allowed extrapolation of certain knowledge obtained in animals. Drosophila melanogaster, studied for decades, has been widely used as model for human diseases as well as to study responses associated with the consumption of several substances. In the present work we explore the impact of tobacco consumption on a model of ‘smoking flies’. Throughout these experiments, we aim to provide information about the effects of tobacco consumption on cardiac physiology. We assessed intracellular calcium handling, a phenomenon underlying cardiac contraction and relaxation. Flies chronically exposed to tobacco smoke exhibited an increased heart rate and alterations in the dynamics of the transient increase of intracellular calcium in myocardial cells. These effects were also evident under acute exposure to nicotine of the heart, in a semi-intact preparation. Moreover, the alpha 1 and 7 subunits of the nicotinic receptors are involved in the heart response to tobacco and nicotine under chronic (in the intact fly) as well as acute exposure (in the semi-intact preparation). The present data elucidate the implication of the intracellular cardiac pathways affected by nicotine on the heart tissue. Based on the probed genetic and physiological similarity between the fly and human heart, cardiac effects exerted by tobacco smoke in Drosophila advances our understanding of the impact of it in the human heart. Additionally, it may also provide information on how nicotine-like substances, e.g. neonicotinoids used as insecticides, affect cardiac function.This article has an associated First Person interview with the first author of the paper.

Cardiac myosin super relaxation (SRX): a perspective on fundamental biology, human disease and therapeutics.

ABSTRACTThe fundamental basis of muscle contraction ‘the sliding filament model’ (Huxley and Niedergerke, 1954; Huxley and Hanson, 1954) and the ‘swinging, tilting crossbridge-sliding filament mechanism’ (Huxley, 1969; Huxley and Brown, 1967) nucleated a field of research that has unearthed the complex and fascinating role of myosin structure in the regulation of contraction. A recently discovered energy conserving state of myosin termed the super relaxed state (SRX) has been observed in filamentous myosins and is central to modulating force production and energy use within the sarcomere. Modulation of myosin function through SRX is a rapidly developing theme in therapeutic development for both cardiovascular disease and infectious disease. Some 70 years after the first discoveries concerning muscular function, modulation of myosin SRX may bring the first myosin targeted small molecule to the clinic, for treating hypertrophic cardiomyopathy (Olivotto et al., 2020). An often monogenic disease HCM afflicts 1 in 500 individuals, and can cause heart failure and sudden cardiac death. Even as we near therapeutic translation, there remain many questions about the governance of muscle function in human health and disease. With this review, we provide a broad overview of contemporary understanding of myosin SRX, and explore the complexities of targeting this myosin state in human disease.This article has an associated Future Leaders to Watch interview with the authors of the paper.

Emergence of the N-Terminal Regulatory Domain of Cardiac Troponin I for Living on Land and the Function of Adult Heart of Higher Vertebrates

The troponin complex plays a central role in regulating the contraction and relaxation of striated muscles. Troponin-based thin filament regulation of muscle contraction emerged approximately 700 million years ago with largely conserved functions. Troponin I (TnI) is the inhibitory subunit of troponin and has evolved into three muscle type-specific isoforms in vertebrates. Cardiac TnI has an N-terminal extension that is specifically expressed in the adult heart, implicating a unique selection value. Cardiac TnI of higher vertebrate species contains beta-adrenergic regulated PKA phosphorylation sites in the N-terminal extension, which is a mechanism to enhance cardiac muscle relaxation and facilitate ventricular filling. Our data mining showed that the N-terminal extension of cardiac TnI first emerged in the genomes of early tetrapods as well as primordial lobe-finned fishes such as the coelacanth whereas it is absent in ray-finned fish. An intriguing finding is the lack of a transitional species that has an N-terminal extension in cardiac TnI without the PKA phosphorylation sites. This apparently rapid evolution of the beta-adrenergic regulation of cardiac function suggests a high selection value in the cardiac function of vertebrate animals on land. To understand the evolution and original function of the N-terminal extension in modulating cardiac muscle contractility, our study comparing the cardiac muscle of primitive lobe-finned fish with that of mammals and ray-finned fish investigates its role in cardiac adaptation to the increased energetic demands of life on land and significance in human health and the treatment of heart failure.

Intravenous Infusion of the β3-Adrenergic Receptor Antagonist APD418 Improves Left Ventricular Systolic Function in Dogs With Systolic Heart Failure

BackgroundUnlike β1- and β2-adrenergic receptors (ARs), β3-AR stimulation inhibits cardiac contractility and relaxation. In the failing left ventricular (LV) myocardium, β3-ARs are upregulated, and can be maladaptive in the setting of decompensation by contributing to LV dysfunction. This study examined the effects of intravenous infusions of the β3-AR antagonist APD418 on cardiovascular function and safety in dogs with systolic heart failure (HF). Methods and ResultsThree separate studies were performed in 21 dogs with coronary microembolization-induced HF (LV ejection fraction [LVEF] of approximately 35%). Studies 1 and 2 (n = 7 dogs each) were APD418 dose escalation studies (dosing range, 0.35–15.00 mg/kg/h) designed to identify an effective dose of APD418 to be used in study 3. Study 3, the sustained efficacy study, (n = 7 dogs) was a 6-hour constant intravenous infusion of APD418 at a dose of 4.224 mg/kg (0.70 mg/kg/h) measuring key hemodynamic endpoints (e.g., EF, cardiac output, the time velocity integral of the mitral inflow velocity waveform representing early filling to time-velocity integral representing left atrial contraction [Ei/Ai]). Studies 1 and 2 showed a dose-dependent increase of LVEF and Ei/Ai, the latter being an index of LV diastolic function. In study 3, infusion of APD418 over 6 hours increased LVEF from 31 ± 1% to 38 ± 1% (P < .05) and increased Ei/Ai from 3.4 ± 0.4 to 4.9 ± 0.5 (P < .05). Vehicle had no effect on the LVEF or Ei/Ai. In study 3, APD418 had no significant effects on the HR or the systemic blood pressure. ConclusionsIntravenous infusions of APD418 in dogs with systolic HF elicit significant positive inotropic and lusitropic effects. These findings support the development of APD418 for the in-hospital treatment of patients with an acute exacerbation of chronic HF.

Abstract 12877: Magnesium Deficiency Causes Reversible Metabolic Diastolic and Systolic Cardiomyopathies

Introduction: Low circulating magnesium (Mg) level is associated with increased cardiovascular mortality, and conversely, dietary Mg intake is associated with a decreased risk of developing heart failure. Hypothesis: We investigated whether Mg deficiency alone could cause cardiomyopathy. Methods: C57BL/6J mice were fed with a low-Mg diet (low-Mg, 15-30 mg/kg Mg) or a normal diet (nl-Mg, 600 mg/kg Mg) for 6 weeks. To test reversibility, half of the low-Mg mice were fed then with normal diet for another 6 weeks (low→nl-Mg group). Results: Mg deficiency increased mortality, especially in female mice. After 6 weeks of low-Mg diet, surviving mice showed significantly decreased serum Mg (0.9±0.1 vs. 2.8±0.1 mg/dL for nl-Mg) and a reciprocal increase in serum Ca, K, and Na. Low-Mg mice exhibited a decreased cardiac ejection fraction (EF%, 39.8±1.9% vs. 52.0±1.7% of nl-Mg) and impaired relaxation (E/e’: 21.1±1.1 vs. 15.4±0.4 of nl-Mg) in echocardiography. At the cellular level, ATP, Ca transient amplitude, sarcoplasmic reticulum (SR) Ca load, resting sarcomere length, and sarcomere shortening were all decreased significantly in low-Mg hearts and cardiomyocytes. These changes were accompanied by evidence of mitochondrial dysfunction with significantly increased mitochondrial ROS production and mitochondrial membrane depolarization. Mg repletion normalized electrolytes, contraction, relaxation, and cellular changes. The SR Ca pump (SERCA) was decreased, the SR Ca channel RyR2 oxidized, and cardiac myosin binding protein C S-glutathionylated in low-Mg mouse hearts. These changes were normalized with Mg repletion. In vivo , mitoTEMPO treatment during low Mg diet improved the cardiac relaxation and increased cellular ATP levels without improving contraction. Conclusions: Mg deficiency caused a reversible diastolic and systolic cardiomyopathy associated with mitochondrial dysfunction. This cardiomyopathy may explain the relationship of hypomagnesemia and worsening heart failure. Mg intake could reverse these changes, reinforcing the known correlation of increased Mg intake and reduced heart failure symptoms and mortality. In deficiency states, Mg supplementation may represent a novel treatment for systolic and diastolic heart failure.

Abstract 12717: Enhance Negative Modulation of Beta 3 -Adrenergic Stimulation on Cardiomyocyte Functional Performance, [Ca 2+ ] i Regulation and β-Adrenergic Reserve in Rats Expressing Human Angiotensinogen Gene

Background: Transgenic rats expressing the human angiotensinogen gene [TGR (hAogen) 1623] (hAogen) exhibit sustained hypertension and systolic dysfunction associated with altered cardiac renin-angiotensin system and β- adrenergic receptor (AR) signal transduction systems. We have shown previously, in contrast to β 1 - and β 2 -ARs, β 3 -ARs are linked to G i proteins, and stimulation of β 3 -ARs inhibits cardiac contraction and relaxation. Hypertension is associated with increased cardiac expression of G i proteins. This may alter β 3 -AR stimulation and play an important role in the cardiac functional impairment of this humanized model of hypertension. However, the alteration and functional effect of β 3 -AR activation in this model are unknown. We tested the hypothesis that high cardiac tissue Ang II content in hAogen may cause an up-regulation of cardiac β 3 -AR, which exacerbates myocyte dysfunction and impairs calcium regulation, thereby directly contributing to the cardiac dysfunction. Methods: We compared LV myocyte β 3 -AR expression and myocyte functional and [Ca 2+ ] i transient ([Ca 2+ ] iT ) responses to β- and β 3 -AR stimulation in myocytes obtained from 8 Sprague Dawley (SD) control and 8 hAogen male adult rats. Results: Versus SD, in hAGT rats, basal myocyte contraction (dL/dt max , 107.1 vs 138.8 μm/s), relaxation (dR/dt max , 94.0 vs 103.9 μm/s) and [Ca 2+ ] iT (0.15 vs 0.21) were depressed. A non-selective β-AR agonist, isoproterenol (ISO) (10 -8 M) produced significantly smaller increases in dL/dt max , (42% vs. 61%), dR / dtmax (35% vs. 51%), and [Ca 2+ ] iT (20% vs. 30%). Moreover, versus SD, in hAogen rats, LV myocyte β 3 -AR protein level increased by 32% (0.29 vs 0.22) with resulted significantly altered myocyte functional response to β 3 -AR stimulation. Compared with the changes in SD myocytes, in hAogen myocytes, BRL (10 -8 M) produced a significantly greater decreases in dL/dt max , (27% vs 12%), dR/dt max (28% vs 11%), and [Ca 2+ ] iT (17% vs 10%). Conclusions: TGR (hAogen) 1623, this humanized model of hypertension is associated with an up-regulation of LV myocyte β 3 -AR, which enhances β 3 -AR-caused inhibition of myocyte contractile, relaxation and [Ca 2+ ] iT and exacerbates β-AR desensitization, thereby directly contributing to the cardiac dysfunction.

Abstract 17492: Chronic Treatment With a Mavacamten-Like Myosin-Modulator (MYK-581) Prevents Left-Atrial Remodeling, Decreases Cardiac Troponin Leakage, and Blunts Mortality in a Mini-Pig Model of Inherited Hypertrophic Cardiomyopathy

Introduction: Hypertrophic cardiomyopathy (HCM) is a progressive disease characterized by cardiac remodeling, impaired relaxation, left-atrial enlargement, and exertional intolerance. Direct myosin-inhibition with mavacamten can normalize contractility and improve exercise capacity in patients with obstructive HCM (oHCM). However, mavacamten also limits residual cross-bridges during diastole, and therefore, may offer cardiac benefits beyond obstruction relief. This in vivo study evaluated the chronic effects of MYK-581, a mavacamten surrogate, in a genetic large-animal model of HCM. Methods: Young cloned Yucatan mini-pigs with a heterozygous MYH7 R403Q mutation were randomly assigned to one of two arms: untreated-controls (CTRL, n = 29) or daily MYK-581 (n = 22; PO); wild-type pigs (WT) untreated served as disease-controls. After 14 weeks of treatment, pigs underwent in vivo cMR imaging, including T1 mapping and extracellular volume (ECV) assessments. In a subset of pigs, biomechanical studies were performed in skinned left-ventricular (LV) and left-atrial (LV) fibers. *, #: P&lt;0.05 vs. CTRL or WT. Results: In HCM pigs, MYK-581 treatment decreased mortality (9.0* vs. 37.9% in CTRL). MYK-581 blunted cTnT leakage, reducing both absolute values (21.2 ± 3.2* vs. 34.0 ± 4.3 ng/L in CTRL) and the incidence of cTnT &gt; 20 ng/L (36* vs. 81% in CTRL). Treated pigs had smaller LA volumes (16 ± 1* vs. 29 ± 4mL in CTRL) with lower LV T1-times and ECV (27 ± 1* vs. 32 ± 2% in CTRL). LA fibers from untreated HCM pigs showed biomechanical remodeling characteristic of chronic overload: increased maximal force (21.8 ± 1.5# vs. 14.7 ± 1.4 mN/mm2 in WT) and slowed cross-bridge formation rates (Ktr: 4.9 ± 0.4 # vs. 6.6 ± 0.6 s-1 in WT) consistent with a switch towards slow-myosin isoforms. MYK-581 prevented this remodeling (e.g., Ktr: 8.1 ± 1.3 s-1*), preserving normal LA (fast) myosin content. Conclusions: Chronic direct myosin attenuation with a mavacamten surrogate prevented left-atrial remodeling, a known prognostic indicator in HCM. Chronic treatment also reduced cardiac troponin leakage characteristic HCM, and, more importantly, decreased mortality. Taken together, these pre-clinical observations show potential salutary effects beyond obstruction relief in HCM.

Effects of Individual and Coexisting Diabetes and Cardiomyopathy on Diastolic Function in Rats (Rattus norvegicus domestica).

The goal of this study was to evaluate diastolic intraventricular pressure gradients (IVPG) and 2-dimensional tissue tracking (2DTT) patterns during diabetes and cardiomyopathy. Rats (n = 60) were induced to become diabetic (DM group, n = 15) by using streptozotocin, to become cardiomyopathic (CM group, n = 15) by using isoproterenol, and to become both diabetic and cardiomyopathic (DMCM group, n = 15); control rats (CT group, n = 15) were injected with saline. Two months after induction, all rats underwent conventional echocardiography, IVPG, and 2DTT and then were euthanized for microscopic examination of cardiac fibrosis. Compared with the controls, all 3 treated groups showed diastolic dysfunction and delayed cardiac relaxation. DMCM rats showed the most pronounced cardiac abnormalities. In addition, CM and DMCM groups had showed decreased middle IVPG, whereas DMCM rats had decreased midapical IVPG. Although the overall IVPG of the CM group was normal, the middle segment was significantly decreased. 2DTT results showed that the DMCM group had a delay in relaxation compared with other groups. IVPG and 2DTT can be used to overcome the limitation of conventional echocardiographic methods and reveal diastolic dysfunction. DM worsened diastolic function during cardiac disease.

Cardioprotection by triiodothyronine following caloric restriction via long noncoding RNAs

Severe caloric-restriction compromises thyroid hormone (TH) status, apparently to save energy and proteins for enduring stress stimulus. However, a persistent decrease in TH levels may compromise heart function. We hypothesized that supplementation of low dose active TH or targeting hypoxia-inducible factor-1-alpha, HIF-1α (a strong activator of deiodinase enzyme that degrades peripheral active THs) will prevent deterioration of cardiac performance. Adult mice were subjected to acute fasting based on institutional animal protocols with ad libitum access to water. The following groups were studied: Control mice with free access to food; severe caloric restriction fasting only group; Fasting with Triiodo-l-Thyronine (T3); Fasting with HIF-1α inhibitor (BAY). Cardiac hemodynamic and electrophysiological studies were performed and role of long noncoding RNAs were explored. Following severe caloric-restriction, we found that body weights, and heart weights to a partial extent, were decreased. Low-dose T3 treatment attenuated left ventricular hemodynamic impairment in indices of cardiac contractility and relaxation. In electrophysiology studies, fasting mice developed atrial tachyarrhythmias upon induction. This reverted to control levels following T3 treatment. There was a significant increase in atrioventricular conduction time and significant decrease in heart rate following fasting. Both these changes were attenuated following T3 treatment. Furthermore, BAY partially improved hemodynamics. Compared to the severe caloric-restriction group, both T3 and BAY reduced MALAT1 and GAS5 long noncoding RNA expression. These new findings indicate that T3 and BAY protect from cardiac decompensation secondary to acute severe caloric-restriction partly mediated by long noncoding RNAs.

Abstract MP135: Cardiac Sarcoplasmic Reticulum Calcium Cycling is Regulated by Kinase Independent Function of Pi3kgamma

Genetic deletion of Phosphoinositide 3-kinase (PI3Kγ) in mice (PI3Kγ -/- ) results in increased cAMP levels and enhanced ventricular contractility. We investigated whether the lack of PI3Kγ plays a role in cardiac contractility by altering intracellular calcium recycling. Isolated cardiomyocytes from PI3Kγ -/- mice showed significantly reduced calcium reuptake by sarcoendoplasmic reticulum (SR) following caffeine induced calcium release indicating that PI3Kγ locally regulates the function of SR. The intracellular calcium remained at elevated levels in the cardiomyocytes of PI3Kγ -/- for a prolonged period after caffeine treatment. This could be due to changes in phosphorylation of SERCA2, Ryanodine receptor (RyR 2 ) or phospholamban (PLN). In fact, when we looked at phosphorylation of PLN in cardiac lysates, a major regulator of cardiac contractility and relaxation, PI3Kγ -/- mice showed significantly reduced PLN phosphorylation compared to littermate controls. Previous studies from our laboratory suggested that absence of PI3Kγ leads to increase in protein phosphatase (PP) activity which could be possible reason for rapid dephosphorylation of PLN, resulting in inhibition of SERCA2 pump. We observed increased SR associated PP activity and PLN associated PP activity in PI3Kγ -/- mice. We also observed increased association of PP-1 and PP2A with PLN in the absence of PI3Kγ. The altered calcium handling in the cardiomyocytes of PI3Kγ -/- mice could be restored to the level of WT controls by okadaic acid mediated inhibition of PP, suggesting that PI3Kγ plays a role in regulating PP activity associated with SR. To test whether PI3Kγ activity is required for PLN dephosphorylation and SR calcium cycling, we used mice with cardiac specific overexpression of kinase dead PI3Kγ (PI3Kγ inact ) in global PI3Kγ -/- mice (PI3Kγ inact /PI3Kγ -/- ). PI3Kγ inact /PI3Kγ -/- mice showed restored PLN phosphorylation, improved caffeine induced calcium reuptake, decreased SR and PLN associated PP activity. These studies show a novel regulation of PP and SR calcium regulation by kinase independent function of PI3Kγ. The underlying mechanism of PP regulation by PI3Kγ will be presented.

Abstract 448: Strain Rate Dependent Myosin Head Position Changes During Relaxation

Impaired cardiac relaxation is present in nearly all cases of heart failure and possibly in up to 25% of the asymptomatic population. Myocardial relaxation is known to be biochemically modified by the calcium reuptake rate, thin filament calcium sensitivity, and crossbridge kinetics. Mechanical regulation of relaxation was thought to be regulated via afterload, but we have recently shown that a lengthening strain was sufficient to modify relaxation. Further, the relaxation rate is actually dependent on the strain rate, a relationship that we termed Mechanical Control of Relaxation. Computational modeling suggests that myosin detachment is a key mechanism underlying Mechanical Control of Regulation, but to date, no experimental evidence for this was available. The objective of this study was to determine if myosin head position changed in response to lengthening strains during relaxation. Intact cardiac trabeculae were mounted within the beamline of the Biophysical Collaborative Access Team (BioCAT) beamline at the Advanced Photon Source at Argonne National Laboratories. The trabeculae were paced and load-clamps were performed during time-resolved imaging of the equatorial axis, which primarily reflects myosin head positioning. Activation (pacing) caused the myosin head localization to shift from the thick filament to near the thin filament (increased I 1,1 /I 1,0 ratio). During stretch, there was a transient decline of the I 1,1 /I 1,0 ratio which recovered until relaxation was complete, when the ratio again reduced indicating myosin returned to the thick filament. These preliminary data suggest that Mechanical Control of Relaxation is caused by perturbations in myosin, but the late-diastolic kinetics suggests that the strain-rate dependent detachment does not lead to immediate deactivation of myosin heads. Modifications of myosin ATPase properties may reveal more specific regulatory targets, which may provide new insight and targets for treating impaired myocardial relaxation.

Transcriptional signatures regulated by TRPC1/C4-mediated Background Ca2+ entry after pressure-overload induced cardiac remodelling

AimsAfter summarizing current concepts for the role of TRPC cation channels in cardiac cells and in processes triggered by mechanical stimuli arising e.g. during pressure overload, we analysed the role of TRPC1 and TRPC4 for background Ca2+ entry (BGCE) and for cardiac pressure overload induced transcriptional remodelling. Methods and resultsMn2+-quench analysis in cardiomyocytes from several Trpc-deficient mice revealed that both TRPC1 and TRPC4 are required for BGCE. Electrically-evoked cell shortening of cardiomyocytes from TRPC1/C4-DKO mice was reduced, whereas parameters of cardiac contractility and relaxation assessed in vivo were unaltered. As pathological cardiac remodelling in mice depends on their genetic background, and the development of cardiac remodelling was found to be reduced in TRPC1/C4-DKO mice on a mixed genetic background, we studied TRPC1/C4-DKO mice on a C57BL6/N genetic background. Cardiac hypertrophy was reduced in those mice after chronic isoproterenol infusion (−51.4%) or after one week of transverse aortic constriction (TAC; −73.0%). This last manoeuvre was preceded by changes in the pressure overload induced transcriptional program as analysed by RNA sequencing. Genes encoding specific collagens, the Mef2 target myomaxin and the gene encoding the mechanosensitive channel Piezo2 were up-regulated after TAC in wild type but not in TRPC1/C4-DKO hearts. ConclusionsDeletion of the TRPC1 and TRPC4 channel proteins protects against development of pathological cardiac hypertrophy independently of the genetic background. To determine if the TRPC1/C4-dependent changes in the pressure overload induced alterations in the transcriptional program causally contribute to cardio-protection needs to be elaborated in future studies.

Alterations in plasma triglycerides and ceramides: links with cardiac function in humans with type 2 diabetes

Cardiac dysfunction in T2D is associated with excessive FA uptake, oxidation, and generation of toxic lipid species by the heart. It is not known whether decreasing lipid delivery to the heart can effect improvement in cardiac function in humans with T2D. Thus, our objective was to test the hypothesis that lowering lipid delivery to the heart would result in evidence of decreased "lipotoxicity," improved cardiac function, and salutary effects on plasma biomarkers of cardiovascular risk. Thus, we performed a double-blind randomized placebo-controlled parallel design study of the effects of 12 weeks of fenofibrate-induced lipid lowering on cardiac function, inflammation, and oxidation biomarkers, and on the ratio of two plasma ceramides, Cer d18:1 (4E) (1OH, 3OH)/24:0 and Cer d18:1 (4E) (1OH, 3OH)/16:0 (i.e., "C24:0/C16:0"), which is associated with decreased risk of cardiac dysfunction and heart failure. Fenofibrate lowered plasma TG and cholesterol but did not improve heart systolic or diastolic function. Fenofibrate treatment lowered the plasma C24:0/C16:0 ceramide ratio and minimally altered oxidative stress markers but did not alter measures of inflammation. Overall, plasma TG lowering correlated with improvement of cardiac relaxation (diastolic function) as measured by tissue Doppler-derived parameter e'. Moreover, lowering the plasma C24:0/C16:0 ceramide ratio was correlated with worse diastolic function. These findings indicate that fenofibrate treatment per se is not sufficient to effect changes in cardiac function; however, decreases in plasma TG may be linked to improved diastolic function. In contrast, decreases in plasma C24:0/C16:0 are linked with worsening cardiac function.